Shop All Pathway Analysis Cell Based Assay Kits

CellSensor™ GAS-bla ME-180 Cell Line

The CellSensor® GAS-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the Gamma Activated Sequence (GAS) response element stably integrated into ME-180 cells. To construct this cell line, the GAS-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interferon Gamma (IFN-γ). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z'and EC50 concentrations for IFN-&gamma. The CellSensor® GAS-bla ME-180 cell line is responsive to IFN-γ and can be used to probe the JAK/STAT signaling pathway. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ SIE-bla THP-1 Cell Line

The CellSensor® SIE-bla THP-1 cell line contains a beta-lactamase reporter gene under control of the Sis Inducible Element (SIE), stably integrated into THP-1 cells. To construct this cell line, the SIE-bla construct was transduced into THP-1 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interferon Gamma (IFNγ). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z´ and EC50 concentrations for IFN&gamma. The CellSensor® SIE-bla THP-1 cell line is responsive to IFNγ and can be used to analyze the JAK/STAT pathway using the GeneBLAzer® Loading Substrates. Academic and non-profit customers, please inquire for special pricing.

BacMam ERK2 [pThr185/pTyr187] Cellular Assay Kit (Invitrogen™)

The BacMam ERK2 [pThr185⁄pTyr187] Cellular Assay Kit is a high-throughput screening (HTS) compatible cellular immunoassay for the measuring of phosphorylation of GFP-ERK2. The BacMam ERK2 Cellular Assay Kit can be used with numerous transformed cell lines, primary cells, and stem cells, and allows for the interrogation of ERK2, either as a kinase or GPCR readout.

• Choose cell types that are relevant to your needs
• Get results in weeks instead of months
• Perform Assays on Your Schedule

Use Cell Types Relevant to Your Needs
Understanding signaling in physiologically relevant cell backgrounds is extremely important when predicting the real effects of compounds in humans prior to clinical trials. With the BacMam ERK2 [pThr185⁄pTyr187] Cellular Assay Kit you can choose the appropriate cell type for your research needs, enabling you to design your own experiments, and to reach your research goals faster.

BacMam ERK2 Reagent Compatible Cells
The BacMam reagent included in the kit has been used to successfully transduce numerous transformed cell lines (e.g. U2-OS, HEK 293, and HeLa), primary cells (e.g., fibroblasts, hepatocytes, cardiovascular cells and epithelial cells), and stem cells (e.g. neuronal and mesenchymal) at high transduction efficiency and low levels of cytotoxicity. However, cells of hematopoietic origin show consistently poor transduction by BacMam technology, and are not recommended for use with the current technology.

Develop Assays Faster
The BacMam platform enables transient target expression allowing for reduced time to an HTS ready assay versus traditional stable cell line creation. Traditional stable cell line development requires months of culturing, optimization, and cloning of your cell line. This requires considerable time and resources. With BacMam ERK2 a user can get results in weeks versus months.

Cells Ready When You Are
BacMam Erk2 Reagent is ideal to transduce large quantities of cells in batch mode; transduced cells can be stored frozen in aliquots for later use. This saves you the delays associated with culturing cells. The cells are ready whenever you are. Cells can be assayed typically within hours of thawing, providing assay-ready cells when you need them, with no loss of activity.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

CellSensor™ TrkA-NFAT-bla CHO-K1 Cell Line

Neurotrophins (NGF, BDNF, NT-3, and NT-4) and their transmembrane receptors (TrkA, TrkB, TrkC, and P75NTR) play important roles in the regulation of neuronal and non-neuronal cell proliferation, differentiation, survival, and death. Neurotrophin signaling also mediates neuronal higher-order activities, such as learning, memory, and behavior. Alterations in neurotrophin levels and their receptors have been implicated in neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders. Neurotrophins propagate their signal by activating multiple signaling pathways. One of the signaling pathways of NGF (the ligand for TrkA) activates phospholipase C, releasing DAG and IP3, increasing downstream intracellular calcium, and activating protein kinase C. This promotes the translocation of the transcription factor, nuclear factor of activated T-cells (NFAT), from the cytosol into the nucleus, resulting in NFAT-dependent transcription.

The CellSensor® TrkA-NFAT-bla CHO-K1 Cell Line was engineered by integrating the human TrkA expression plasmid into the genome of existing CellSensor® NFAT-bla CHO-K1 Cell Line, which is engineered to express beta-lactamase under the control of NFAT. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and in cryopreserved cells, and has been validated for Z´-factor and EC50 values under optimized conditions using NGF 2.5s.

Additional testing information using various small molecule inhibitors is also provided.

CellSensor™ irf1-bla CTLL-2 Cell Line

Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of the Jak/Stat pathways, Jak2/Stat5, mediates cell proliferation in response to Interleukin - 3 (IL - 3), prolactin, erythropoietin (Epo), and granulocyte - macrophage colony stimulating factor (GM - CSF). JAK2 gene knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation. The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds to a specific palindromic DNA sequence found in the promoter region of β - casein, interferon regulatory factor - 1 (irf - 1) and a number of other genes. The CellSensor® irf1 - bla CTLL2 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into CTLL2 cells. CTLL2 cells are a clone of cytotoxic T cells derived from a C57BL/6 mouse, and are cell - growth dependent on mouse Interleukin - 2 (mIL - 2). This cell line is validated for EC50 and Z' - factor under optimized conditions using mIL - 2. This cell line has also been tested under variable assay conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to pathway inhibitors was also tested. CellSensor® irf1 - bla CTLL2 cells were stimulated with mIL - 2 in triplicate over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios for each replicate plotted against the indicated concentrations of mIL - 2. CellSensor® irf1 - bla CTLL2 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format for 30 min. Cells were then incubated for 5 hrs with mIL - 2 agonist (1 ng/ml) in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader, converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 stimulated cells), and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ NFAT-bla Jurkat Cell Line

The CellSensor® NFAT-bla Jurkat cell line contains a beta-lactamase reporter gene under control of a Nuclear Factor of Activated T Cells (NFAT) response element stably integrated into Jurkat cells. This cell line can be used as a parental cell line to build specific G-protein coupled receptor (GPCR) assays after transfection of additional genes of interest. NFAT-bla Jurkat cells have been shown to be responsive to phorbol 12-myristate 13-acetate (PMA) and Ionomycin stimulation. Additional testing data using alternate stimuli is available upon request. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ HRE-bla HCT-116 Cell Line

Hypoxia is an almost universal hallmark of solid tumors. Adaptation to hypoxia is critical for tumor survival and growth, and is mediated largely by transcriptional activation of genes that facilitate short - (e.g., glucose transport) and long - term (e.g., angiogenesis) adaptive mechanisms. This coordinated homeostatic response is mediated in large part through the activation of the heterodimeric transcription factor hypoxia - inducible factor 1 (HIF - 1). Under conditions of normal oxygenation, the regulated HIF - 1α is hydroxylated and degraded by the proteasome system. As oxygen becomes rate limiting, hydroxylation diminishes and HIF - 1α accumulates and heterodimerizes with the constitutively present α - subunit. The binding of this complex to the cognate hypoxia - response element (HRE) results in transcriptional activation of genes containing such elements within promoter or enhancer elements. The CellSensor® HRE-bla HCT - 116 Cell Line contains a beta - lactamase reporter gene under control of the HRE stably integrated into HCT - 116 cells. This cell line is validated for EC50 and Z'; - factor under optimized conditions using deferoxamine (DFO) and Cobalt Chloride. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. CellSensor ® HRE-bla HCT - 116 cells were stimulated with deferoxamine (DFO) over the indicated concentration range in a 384 - well format. Cells were incubated for 24 hrs with agonist and 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The Response Ratios were plotted against the indicated concentrations of DFO. CellSensor® HRE - bla HCT - 116 cells were stimulated with Cobalt Chloride (CoCl2)over the indicated concentration range in a 384 - well format. Cells were incubated for 24 hrs with agonist and 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. Response ratios were plotted against the indicated concentrations of CoCl2. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ irf1-bla Ba/F3 Cell Line

Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of the Jak/Stat pathways, Jak2/Stat5, mediates cell proliferation in response to Interleukin - 3 (IL - 3), prolactin, erythropoietin (Epo), and granulocyte - macrophage colony stimulating factor (GM - CSF). JAK2 gene knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation. The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds to a specific palindromic DNA sequence found in the promoter region of β - casein, interferon regulatory factor - 1 (irf - 1), and a number of other genes. The CellSensor® irf1 - bla BA/F3 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into BA/F3 cells. This cell line is validated for EC50 and Z' - factor under optimized conditions using mouse IL - 3 (mIL - 3). This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to Jak Inhibitor 1, a small molecule inhibitor, was also tested. CellSensor® irf1 - bla BA/F3 cells were treated with mIL - 3 in triplicate over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with mIL - 3 agonist in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The Response Ratios for each replicate were plotted against the indicated concentrations of mIL - 3. CellSensor ® irf1 - bla BA/F3 cells were treated with Jak Inhibitor 1 for 30 min over the indicated concentration range in a 384 - well format. Cells were then incubated with mIL - 3 agonist in 0.5% DMSO for 5 hrs and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The emission ratios were plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

BacMam Histone H3 [AcLys9] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of histones. BacMam provides a convenient genetic delivery tool for a GFP-Histone H3 fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring acetylation of GFP-Histone H3 at Lys9.

CellSensor™ HRE-bla ME-180 Cell Line

The CellSensor® HRE-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the hypoxia response element (HRE) stably integrated into ME-180 cells. To construct this cell line, the HRE-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to deferoxamine. This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations for cobalt chloride and deferoxamine. The CellSensor® HRE-bla ME-180 cell line is responsive to deferoxamine and cobalt chloride and can be used to probe the hypoxia signaling pathway. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ Myc-bla HCT116 Cell Line

The CellSensor™Myc-bla HCT116 cell line contains a beta-lactamase reporter gene under the control of Myc binding sequences. The construct was transduced into HCT116 cells using a lentiviral system. HCT116 is a colon cancer cell line which expresses a mutated form of beta-catenin. This form of beta-catenin leads to the accumulation of beta-catenin and constitutive activation of downstream genes such as Myc. This cell line is a clonal population isolated by flow cytometry. It has been validated for cell plating density and DMSO tolerance. The signaling pathway has been validated using RNAi against c-Myc and ICG-001, an inhibitor of the wnt-beta-catenin pathway. The expression of the mutant beta-catenin in HCT116 cells results in constitutive activation of beta-lactamase in this CellSensor™line, which can be knocked down by ICG-001 (Figure 1) or Myc RNAi (Figure 2).

CellSensor™ c-Fos-bla HEK293T Cell Line

The c-fos-bla HEK 293T cell line contains a beta-lactamase reporter gene under control of the c-fos promoter stably integrated into HEK 293T cells. This cell line can be used to detect agonists/antagonists of the c-fos pathway. The c-fos-bla HEK 293T cells have been shown to be responsive to phorbol 12-myristate 13-acetate (PMA) stimulation. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ ISRE-bla HEK293T Cell Line

Interferon cell signaling is a critical pathway involved in viral defense and autoimmune disease. The CellSensor® ISRE-bla HEK 293T cell line is responsive to interferon alpha and can be used to probe the JAK/STAT signaling pathway.The CellSensor™ ISRE-bla HEK 293T cell line contains the β-lactamase reporter gene under the control of the interferon stimulated response element (ISRE). This cell line was engineered by transduction of the ISRE-bla construct into HEK 293T cells by lentivirus. Flow cytometry was then used to isolate a pool of clones responsive to interferon alpha a stimulation. A dose response curve for this assay against interferon alpha is available in the validation packet. Academic and non-profit customers, please inquire for special pricing.

BacMam Histone H3K4me3 Cellular Assay Kit (Invitrogen™)

The BacMam Histone H3K4me3 Cellular Assay is a high-throughput screening (HTS) compatible TR-FRET assay for the interrogation of trimethylation at lysine 4 on histone H3 in a cellular format. Baculovirus-mediated gene delivery (BacMam) provides a convenient tool for the expression of GFP-Histone H3 fusion protein in your cell background of interest. Coupled with a terbium (Tb)-labeled site-specific antibody, the resulting cellular assay can be used to identify inhibitors of methyltransferases and demethylases acting on lysine 4 of histone H3.

The kit includes:

• BacMam Histone H3 Reagent
• LanthaScreen® Tb-anti-histone H3K4me3 Antibody
• 6X LanthaScreen® Cellular Lysis Buffer
• Instrument Control Terbium TR-FRET kit

The BacMam Histone H3K4me3 Cellular Assay Kit Lets You:
• Use your cell background of choice with the portability of BacMam
• Identify more relevant inhibitors since the methyl transferases and demethylases are in their natural protein complexes
• Conserve precious cell samples with the miniaturizable homogenous assay format
• Improve data quality with the advantages of TR-FRET

Get More Physiologically Relevant Results
The BacMam Histone H3K4me3 Cellular Assay allows the investigation of trimethylation at lysine 4 on histone H3 with your choice of cellular backgrounds including primary cells. This enables screening for potential inhibitors of methyl transferases and demethylases associated with Lys4 trimethylation in their natural complexes in a physiologically relevant cell type. Please note that the LanthaScreen® Tb-anti-Histone H3K4me3 antibody has been shown to have higher affinity for trimethylation compared to dimethylation at lysine 4 using a histone H3 peptide; however, when interpreting your results, it is important to consider that the antibody does show some affinity for the dimethylated peptide. Further discrimination between di- and trimethylation can be accomplished by using our complimentary BacMam Histone H3K4me2 Cellular Assay Kit (Cat # A14164), which is specific for dimethylation.

BacMam Convenience Saves Samples and Time
Assays are run in a fully homogenous, addition-only format without any of the washing, lysate transfer, or separation procedures required for traditional methods such as Western blotting and ELISA, making them ideal for HTS applications. By using the same BacMam reagent for GFP-Histone H3, other histone H3 modifications (Ph, Me, Ac) can be monitored just be exchanging the Terbium (Tb) labeled antibody. In addition, the application of the LanthaScreen® technology includes all the advantages of TR-FRET detection including reduced data noise, less interference from fluorescent compounds, and high sensitivity, allowing the use of fewer cells than traditional Western or ELISA methods.

For research use only. Not for human or animal therapeutic or diagnostic use.

CellSensor™ TNFa-bla THP-1 Cell Line

Tumor necrosis factor-alpha (TNFα) cytokine cell signaling is a critical pathway involved in immune and inflammatory responses. In addition, TNFα has been shown to inhibit growth and induce differentiation in tumor and non-tumor cells. The CellSensor™ TNFα-bla THP-1 cell line is a suspension cell line derived from human acute monocytic leukemia cells. The TNFα-bla THP-1 cell line is used to measure the TNFα cytokine signaling pathway via transcriptional regulation of the beta-lactamase gene. This cell line was constructed using GenomeScreen™ technology utilizing a promoterless beta-lactamase reporter gene randomly integrated into the genome of the THP-1 cell line. Sequential rounds of sorting were performed to isolate cell clones containing tagged genes induced by TNFα. These cells have been shown to be responsive to TNFa agonist. Academic and non-profit customers, please inquire for special pricing.

BacMam Akt [pSer473] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Ser473.

CellSensor™ NFAT-bla CHO-K1 Cell Line

The G protein-coupled receptor (GPCR) superfamily is comprised of an estimated 600-1,000 members and is the largest known class of molecular targets with proven therapeutic value. GPCRs are instrumental in the transmission of a wide range of chemical messages from the extracellular environment to the interior of the cell. GPCRs are involved in a wide range of disorders, including allergies, cardiovascular dysfunction, depression, obesity, cancer, pain, diabetes, and various central nervous system disorders. GPCR signaling is mediated by trimeric G-proteins containing α, β, and γ subunits and can be categorized into signaling classes based upon subunit composition. G alpha, G alpha s, and G alpha i/o proteins mediate intracellular signaling through activation of signaling pathways leading to distinct physiological endpoints. Activation of G alpha s and G alpha i/o coupled receptors leads to stimulation or inhibition of adenylate cyclase, respectively; activation of G alpha q coupled receptors results in stimulation of phospholipase C. GPCR signaling through these distinct pathways can be monitored by activation of specific transcriptional response elements placed upstream of a reporter gene. Invitrogen has developed GeneBLAzer® Master Cell Lines employing the beta-lactamase reporter system, which can be used to screen for both agonists and antagonists of GPCRs coupling to each of the above signaling pathways. These cell-based assays make use of a membrane permeant fluorescent substrate (CCF4-AM or CCF2-AM), enabling a ratiometric readout that reduces errors due to sample variability, leads to excellent Z'-factor values, and allows for assay miniaturization. GeneBLAzer® Master Cell Lines with either NFAT or CRE response elements are available in Jurkat, CHO-K1, or FreeStyle™ 293F cell backgrounds (six cell lines in total) for the development of assays in suspension or adherent cell format. Stable cell lines employing an NFAT response element upstream of the beta-lactamase gene are designed for the study of Gq coupled receptors. Stable cell lines employing the cAMP response element (CRE) upstream of the beta-lactamase gene are designed for the study of Gs coupled receptors. FreeStyle™ 293F cells are HEK 293 cells that have been adapted for growth in suspension in serum-free media. Alternatively, these cells may be grown in the presence of serum for use in the adherent format. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ IL1-bla ECV304 Cell Line

The CellSensor® IL-1-bla ECV304 cell line is an adherent human urinary bladder cell line used to measure the (IL-1) signaling pathway via transcriptional regulation of the beta-lactamase gene. IL-1 cytokine cell signaling is a critical pathway involved in immune and inflammatory responses. This cell line was constructed using GenomeScreen™ technology utilizing a promoterless beta-lactamase reporter gene randomly integrated into the genome of the IL-1-bla ECV304 cell line. Sequential rounds of sorting were performed to isolate cell clones containing tagged genes induced by IL-1. A dose response assay was run with this cell line using IL-1 as the agonist. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ T-REx™ FOXO3 DBE-bla HeLa Cell Line

The CellSensor® T-REx™ FOXO3 DBE-bla HeLa cell line was engineered by lentiviral transduction of HeLa cervical cancer cells with a FOXO3 response element driving beta-lactamase expression (DBE-bla), along with a tetracycline repressor and tetracycline (or the tetracycline analog doxycycline)-inducible FOXO3 constructs. Addition of doxycycline to these cells allows for FOXO3 transcription factor expression and subsequent beta-lactamase expression, which in turn can be down-regulated by insulin-mediated activation of the PI3K/AKT signaling pathway which leads to phosphorylation and inactivation of FOXO3 and concomitant suppression of beta-lactamase expression. This cell line has been tested for robust assay performance by assessing a variety of assay parameters, including: cell plating number, DMSO tolerance, stimulation time, and substrate loading time. Assay validation was performed using serial dilutions of insulin to antagonize doxycycline- induced FOXO3 activity (Figure 1). Additional testing data using RNAi and alternate stimuli are also available.

CellSensor™ AP-1-bla A375 Cell Line

The CellSensor® AP1-bla A375 cell line contains a beta-lactamase reporter gene under control of the AP1 response element stably integrated into A375 cells. A375 cells are human melanoma cancer cells that contain the endogenous B-Raf mutation V600E resulting in constitutive B-Raf kinase activity. The CellSensor® AP1-bla A375 cell line is a clonal population isolated by flow cytometry based on constitutive expression of beta-lactamase. This cell line has been validated with various small-molecule Raf inhibitors as well as B-Raf Stealth RNAi™ siRNA. This cell line has also been tested for assay performance under variable conditions, including DMSO concentration, cell number, compound incubation time, and substrate loading time and validated for Zfi-factor and IC50 concentrations of Raf1 Inhibitor I. The RAF gene family (RAF1, A-RAF, and B-RAF) encodes closely related serine/threonine protein kinases that are important effectors of Ras activation. Raf1 and A-Raf are rarely mutated, whereas mutations in B-Raf are common in human cancers, especially melanoma. B-Raf is mutated in about 70% of human melanomas, 35-70% of papillary thyroid carcinomas, and less commonly in lung and colorectal carcinomas. Mutations are mostly in the B-Raf kinase domain and, in melanomas, the vast majority are V600E missense mutations leading to activation of B-Raf kinase. The constitutive activity of B-Raf V600E can directly lead to the activation of the Mek/MAPK signaling pathway. Therefore, inhibition of B-Raf/Mek/MAPK signaling could be a potential way of treating melanomas and other tumors with mutant B-Raf.

CellSensor™ ISRE-bla Jurkat Cell Line

The CellSensor® ISRE-bla Jurkat cell line contains a beta-lactamase reporter gene under control of the interferon-stimulated response element (ISRE) stably integrated into Jurkat cells. To construct this cell line, the ISRE-bla construct was transduced into Jurkat cells by lentivirus. Flow cytometry was used to isolate cells responsive to IFNα. This cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as ' and EC50 concentrations of IFN&alpha. The CellSensor® ISRE-bla Jurkat cell line is responsive to Interferon alpha (IFN-alpha) and Interferon beta and can be used to probe the type I interferon-induced JAK-STAT signaling pathways. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can also be tested for dose response against this cell line. ISRE-bla Jurkat cells were treated with agonist IFN-alpha; over the indicated concentration range in a 384-well format. Cells were incubated for 5 hours with agonist and 0.5% DMSO and then combined with LiveBLAzer™-FRET B/G Substrate (CCF4-AM) for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader, and the 460/530 ratios were plotted against the concentration of the agonist. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ C/EBP-bla FreeStyle™ 293F Cell Line

CCAAT⁄enhancer-binding protein (C⁄EBPa basic leucine zipper transcription factor, is involved in the regulation of mitotic growth arrest and differentiation. Mutations in C⁄EBPare thought to result in diseases such as human acute myeloid leukemia. C⁄EBPis expressed in liver, fat, lung, peripheral leukocytes, epidermis, intestine, and skeletal muscle. C⁄EBPalso plays a role in energy homeostasis, whereas dominant negative mutations cause altered hepatic glucose and glycogen metabolism as well as defects in white adipose tissue differentiation. The CellSensor® C⁄EBP-bla Freestyle™ 293F cell line contains a beta-lactamase reporter gene under control of the C⁄EBP response element stably integrated into Freestyle™ 293F cells. To construct this cell line, the C⁄EBP-bla construct was transduced into Freestyle™ 293F cells by lentivirus. Flow cytometry was used to isolate cells responsive to Phorbol 12-Myristate 13-Acetate (PMA). This cell line was tested under various conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and functionally validated for Z’ and EC50 concentrations using PMA. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ NFkB-bla RAW 264.7 Cell Line

The CellSensor® NFκB-bla RAW264.7 mouse macrophage cell line contains a beta-lactamase reporter gene under control of a stably integrated NFκB response element. This cell line is a clonal population isolated in response to lipopolysaccharide by flow cytometry. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time, and has been validated for Zfi-factor and EC50 concentrations of the TLR7 ligand imiquimod. Additional data using alternate stimuli are shown. The molecules responsible for coordinating the immune systems recognition of pathogens are Toll-like receptors (TLRs), of which there are currently 10 identified members in humans. TLRs are pattern recognition receptors (PRRs), which bind to pathogen-associated molecular patterns (PAMPs), small molecular sequences consistently found on pathogens. This binding activates the transcription of immune system genes and regulators such as cytokines, chemokines, and costimulatory molecules, thereby promoting the initial immune response of macrophages and neutrophils. Academic and non-profit customers, please inquire for special pricing.

GeneBLAzer™ Jurkat Control Kit

Th e GeneBLAzer® Jurkat Control Kit contains constitutively expressing (CMV-bla) and wildtype Jurkat cells to provide positive and negative controls, respectively, for evaluating beta-lactamase activity (Figure 1).

LanthaScreen™ IGF-1R GripTite™ Cell Line

The IGF-1R⁄PI3K⁄AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake. IGF-1R is a receptor tyrosine kinase (RTK) that resides at the top of the pathway. Upon growth factor binding extracellularly, this RTK undergoes auto-phosphorylation at multiple intracellular tyrosine residues (resulting in pathway activation). The LanthaScreen® IGF-1R GripTite™ (HEK 293 MSR) cellular assay utilizes a human cell line that constitutively expresses IGF-1R fusion proteins with yellow fluorescent protein (YFP). This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using YFP fluorescence as a sorting marker and Blasticidin to maintain cells under selection. Using this cell line, a homogenous immunoassay was developed with a time-resolved FRET (TR-FRET) readout in which the insulin-induced phosphorylation of multiple tyrosine residues on IGF-1R is detected in cell lysates using a generic terbium-labeled phosphospecific antibody (Tb-anti-pY20). This cell line has been validated with different stimuli⁄inhibitors and shows correct EC50 ⁄ IC50 values. Moreover, this assay has been optimized for performance under variable experimental conditions (including cell plating density, agonist stimulation time, DMSO tolerance and assay development time) and displays excellent statistical data (Z' > 0.6) and good signal-to-background.

BacMam Akt [pThr308] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Thr308.

LanthaScreen™ PDCD4 HEK 293E Cell Line

The PI3K⁄AKT⁄mTOR pathway mediates signals for cell growth and survival, transcription and translation, and regulated cell death. The significance of mTOR has been underscored recently by the identification of two distinct cellular complexes: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to rapamycin). Activation of the pathway by insulin (or other mitogens) leads to the phosphorylation of programmed cell death protein 4 (PDCD4) via mTORC1 (indirectly through the ribosomal protein p70 S6 kinase). LanthaScreen® PDCD4 HEK293E is a human cell line which constitutively expresses GFP-PDCD4 fusion proteins. This kinase target was introduced using lipid transfection followed by selection with Blasticidin. These cells are a clonal population isolated by FACS using GFP fluorescence as a sorting marker. A homogenous immunoassay ("addition only", no wash steps) was developed in 384-well format using a TR FRET-based detection method to monitor the phosphorylation of Ser457 on GFP-PDCD4 using a terbium-labeled phosphospecific antibody. The assay has been optimized for performance under numerous experimental conditions (e.g., cell plating density, DMSO tolerance, insulin stimulation time, etc.) and displays excellent statistical data (Z' > 0.6). Moreover, this cell line has been validated with different stimuli and known inhibitors (shows correct EC50 and IC50 values) and serves as robust cellular readout for mTORC1 signaling.

CellSensor™ LEF/TCF-bla HCT-116 Cell Line

Wnt signaling via Beta-catenin plays a central role in development and homeostasis. This pathway is invariably disrupted in colorectal tumors and commonly affected by mutation in other cancers. Wnt ligand binding and activating the Frizzled transmembrane receptors transduced the signal to a cytoplasmic protein, known as disheveled protein, which then inhibits the serine/threonine kinase Glycogen Synthase-3 Beta (GSK-3B). This signal leads to functional inactivation and dissociation of a multi-protein Beta -catenin destruction comples, which is made up of the tumor suppressor protein Adenomatous Polyposis Coli (APC), GSK-3B and a scaffold of Axin. This results in dephosphorylation and dissociation of Beta -catenin. The unphosphorylated b-catenin is stabilized and accumulates in the cytoplasm of the cell. Beta -catenin then associates with the T-Cell Factor (TCF)/Lymphoid Enhancer Factor (LEF) family of transcription factors in the nucleus leading to transcription and expression of target genes, such as c-Myc, c-jun, Fra and cyclin D1. The CellSensor™ LEF/TCF-bla HCT-116 cell line contains a beta-lactamase reporter gene under control of the LEF/TCF stably integrated into HCT-116 cells. HCT-116 is a colon cancer cell line carrying a gain-of-function mutation in beta-catenin gene (deletion of amino acid Serine45), which prevents beta-catenin protein degradation leading to constitutive activation of downstream genes. Thus, this cell line constitutively expresses beta-lactamase, which may be further stimulated or inhibited, e.g., using Stealth RNAi™ siRNA. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Academic and non-profit customers, please inquire for special pricing.

BacMam Aequorin Kit (Invitrogen™)

The BacMam Aequorin Kit utilizes a modified baculovirus (insect cell virus with a mammalian promoter) to transiently deliver aequorin, a luminescent calcium sensor, into mammalian cells from a wide variety of sources: cell lines, primary cells, and stem cells. Upon binding calcium ions, aequorin displays a "flash" style luminescent signal while consuming coelenterazine as a substrate. The luminescence signal resolves problems associated with autofluorescent compounds in traditional assays and provides an exceptional signal-to-noise ratio.

The BacMam Aequorin Kit Allows You to:

Measure calcium in a wide variety cell types

• Develop an assay in 2-3 weeks rather than months
• Take advantage of a large assay window
• Avoid false positives due to autofluorescent compounds

Study Signaling in the Most Relevant Cellular Background
Understanding signaling in physiologically relevant cell backgrounds is extremely important when predicting the real effects of compounds in humans prior to clinical trials. With the BacMam Aequorin Kit you can choose the appropriate cell type for your research needs, enabling you to design your own experiments and to reach your research goals faster.

Save Time and Money with Faster Assay Development
The BacMam Aequorin Kit enables transient target expression allowing for reduced time to an HTS-ready assay versus traditional stable cell line creation. Traditional stable cell line development requires months of culturing, optimization, and cloning of your cell line. This requires considerable time and resources. With BacMam Aequorin a user can get results in 2-3 weeks versus months.

Design the Assay to Meet Your Requirements
The BacMam Aequorin Kit provides flexibility to your research, allowing you to understand signaling across various cell backgrounds. BacMam Aequorin can interrogate calcium signaling in both GPCR and Ion Channel targets whether endogenously or exogenously introduced. To gain even greater flexibility, transduce large quantities of cells in batch mode and store frozen transduced cells in aliquots to use at your convenience.

Obtain High Quality Results with a Large Dynamic Range
BacMam Aequorin is a luminescent calcium indicator. Luminescence readouts present little to no background signal, increasing the robustness of the assay; and they have a large assay window. The assay uses luminescence, thereby minimizing the number of false positive hits due to compound autofluorescence.

BacMam Aequorin Kit Compatible Cells
The BacMam Aequorin Kit has been used to successfully transduce numerous transformed cell lines (e.g. U2-OS, HEK 293, CHO-K1 and HeLa), primary cells (e.g., fibroblasts, HASMC and HUVEC), and stem cells (e.g. neuronal and mesenchymal) at high transduction efficiency and low levels of cytotoxicity. However, cells of hematopoietic origin show consistently poor transduction by BacMam technology, and are not recommended for use.

For research use only. Not intended for human or animal therapeutic or diagnostic use.

CellSensor™ CRE-bla CHO K1 Cell Line

The CellSensor® CRE-bla CHO-K1 cell line contains a beta-lactamase reporter gene under control of a cAMP response element (CRE) stably integrated into CHO-K1 cells. This cell line can be used as a parental cell line to build specific G-protein coupled receptor (GPCR) assays after transfection of additional genes of interest or to detect changes in intracellular cAMP levels. CRE-bla CHO-K1 cells have been shown to be responsive to forskolin stimulation. Academic and non-profit customers, please inquire for special pricing.